The neural crest (NC) plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations are craniofacial in nature and mostly due to such NC failures. We have used the frog Xenopus and the freshwater fish Danio rerio(zebrafish)as experimental model organisms to study NC development. The starting point for this project was two transcription factors, TFAP2a and Dlx3, the regulation of which we showed several years ago to be critical for the early steps in NC development. Since then we have identified several target genes for TFAP2a regulation, and have studied these genes to help understand how NC cells form, migrate and differentiate. One interesting TFAP2a target we discovered was the novel gene Inka. While loss of Inka has turned out to be apparently non-phenotypic in both zebrafish and mouse, the Inka protein was shown to interact strongly with the protein kinase PAK4. Thiis has led to an interesting project on PAK4 function in zebrafish, linking maternal expression of PAK4 to several aspects of embryonic development, implicating a form of transcriptional control mediated by the actin cytoskeleton, of which PAK4 is an important regulator. The role of Dlx3 in NC development is biphasic. According to our earlier work with Xenopus, in order for NC induction to occur, Dlx3 must be excluded from NC cells at early stages, being expressed in adjacent, lateral cells. Later, this factor is expressed in NC derivatives, as well as in epidermis, where it is required for terminal differentiation. Mutation of the human Dlx3 gene results in a dominant genetic disease affecting tooth, craniofacial bone and other structures. To learn more about Dlx3 in NC we will express the mutated and also wild-type versions of this gene in zebrafish NC using transgenes controlled by a NC-specific promoter element driving an inducible activator protein. This will allow us to control the level of Dlx3 proteins in NC cells by adding inducer to the fish water. We will then monitor NC development in live embryos by confocal microscopy. Eventually we will use similar approaches to investigate target genes for Dlx3, as we have for TFAP2a, continuing to elucidate the NC control network In parallel with the Dlx3 project, we are using the same inducible transgenic strategy to gain temporospatial control of cell-cell signaling in migrating and postmigratory NC cells. Our primary focus is on bone morphogenetic proteins (BMPs), especially BMP4 which has been implicated in craniofacial patterning in several vertebrate species. Our sophisticated control strategy should reveal further aspects of how this and other signaling pathways regulate the shape of the vertebrate head, face and jaws, which will help in the diagnosis and treatment of NC-based birth defects and disease.